Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the leading arthropodborne infection in the United States and causes significant morbidity in endemic areas. If untreated B. burgdorferi can persistently infect individuals even though the host mounts a potent adaptive immune response such that antibodies obtained from infected patients or experimentally infected animals effectively kills in vitro cultivated B. burgdorferi. In addition, a robust cell-mediated proinflammatory response is observed that induces IL-6, IL-12 and IFN-??and inhibits IL-10. Furthermore, the spirochete can resist complement killing demonstrating that this important component of the innate immune response is not sufficient to eliminate B. burgdorferi infection. The observation that B. burgdorferi persists in such a hostile environment indicates that the spirochete is adept at evading the host immune response via mechanisms that have not been completely elucidated. One possibility is that B. burgdorferi invades host cells and survives at low levels. Recently we have determined that B. burgdorferi invade both immortalized and, more importantly, primary cells (both fibroblasts and endothelial cells) and persist as viable cells in o-culture. In addition we have preliminary data suggesting that the ability to invade host cells involves both integrin binding and Src kinase activity. In this application we propose to further characterize the internalization of B. burgdorferi and track the fate of B. burgdorferi within thes infected cells to determine how they affect the localized host response following infection. To accomplish this we will use both in vitro correlates of invasion and intracellular survival as well as in vivo imaging of experimentally infected mice as readouts for our studies. Specifically, we propose to: (1) Characterize the invasion of Borrelia burgdorferi into primary fibroblasts. The working hypothesis here is that B. burgdorferi exploits invasion as an additional mechanism to avoid host clearance. Our preliminary studies demonstrate that B. burgdorferi invasion is not dependent on host fibronectin, but does involve ?1 integrins other than ?5?1. In this Aim we will identify the ??subunit that pairs with ?1 to promote invasion and will also evaluate how B. burgdorferi traffics within these cells; and (2) Determine if invasion is required for B. burgdorferi persistence in vivo. Our working hypothesis is that invasion contributes to persistence by providing an immunoprotected niche for B. burgdorferi. Since Src kinases are required for borrelial internalization in vitro, we will determine whether Src kinase inhibitors aler the infectivity potential of B. burgdorferi in vivo. In addition to standard cultivation and molecuar approaches, novel in vivo imaging will be employed to assess how the inhibitor affects colonization. The overall goal of these studies is to determine the extent in which an intracellula locale contributes to borrelial persistence.